The present invention relates to a method of stabilizing enzymic solutions useful for determining urea. Such stabilized solutions have utility in determining the quantitative amount of urea in human sera, such as blood, plasma and the like. More particularly, the invention relates to stabilized enzymic solutions for use in methods for determining urea based upon conductivity measurements.
A method for determining urea was reported by W. T. Chin and W. Kroontje, "Conductivity Method for Determination of Urea," Anal. Chem., 33:1757-60 (1961). The method of Chin and Kroontje was based upon the difference in electrical conductivity of urea and ammonium carbonate produced by the reaction of urease with urea in solution. The method of Chin and Kroontje was modified by P. Bourrelly and V. Bourrelly-Durand, J. Chim. Phys., 62:673-77 (1965) and by M. Hanss and A. Rey, Biochim. Biophys. Acta., 227:630-38 (1971).
An improvement in the method of measuring urea quantitatively using conductivity measurements was developed by G. Paulson, R. Roy and J. Sternberg, "A Rate-Sensing Approach to Urea Measurement," Clin. Chem., 17:644 (1971). The improved method was based on conductivimetric rate-sensing. The method employed the rate of increase of conductivity of a solution as urease catalyzes the hydrolysis of urea to ammonium carbonate by the reaction: ##STR1## The observed rate of change of conductivity was shown to be proportional to the urea concentration in the sample. In particular, the rate of change during the interval from 10 to 20 seconds following addition of the sample to a buffered urease solution at a pH 7 and temperature of 34.degree. C. was determined to be proportional to the urea concentration. The rate-sensing method is sufficiently precise and accurate and has found routine use in clinical chemistry laboratories.
Other methods for quantitative measuring of urea are based on colorimetric and spectrophotometric analyses. For example, in the reaction sequence: ##STR2## the production of ammonia or CO.sub.2 can be quantitatively measured photometrically. The reaction sequence (2) above is the same as reaction sequence (1) except that (2) is written as if it were a nonaqueous system to show that ammonia and carbon dioxide are products of urease activity in urea. The ammonia produced can be measured photometrically by the reaction: EQU NH.sub.3 +4-aminoantipyrine+NaOC1.fwdarw.blue color (3)
The intensity of the blue color produced can be correlated to the amount of ammonia. The ammonia can also be measured through the following reaction sequence: ##STR3## In this reaction sequence the NADH acts as a reducing agent. The NADH has an absorbance at 340 nm and the NAD does not. Thus, the absorbance at 340 nm can be correlated to the amount of NH.sub.3 present in a sample.
Stability of enzymic solutions used in diagnostic assays is important in providing methods of analysis which exhibit precision and uniformity among separate determinations when conducted over a period of elapsed time. Instability of enzymic solutions, in addition to not providing reproducibility of assays, can also add to the ever increasing cost of medical services because the unstable enzymic solutions need to be discarded and fresh solutions formulated.
It has recently been estimated that about 25% of all in vitro diagnostic tests conducted annually in the United States, are unreliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement derives from the fact that the exact nature of enzymes, as well as mechanisms of their reactions, remains unknown for the most part.
At present, the greatest limitation in the diagnostic reagent manufacture, by far, lies in the unstable characteristics of the enzymic solutions. Current urea enzymic diagnostic methodologies require the use of labile ingredients. Due to the labile nature of the enzymes, rigorous quality control is required over the production of such enzymic solutions, in reconstituting dry media preparations and formulation of such enzymic solutions. Such quality control is costly. Moreover, if such control in any step in the process is not maintained within a high degree of control standards, the quality of the final product can be reduced materially leading to decreased precision in assay results.
The present commercial state-of-the-art technique for stabilizing the reactive ability of enzymes or coenzymes is performed by locking them into a solid matrix, either by freeze-drying, dry blending such as used for tableting dry powders primarily in the pharmaceutical diagnostic and related industries, and immobilization by locking the chemical structure of the enzyme into a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze-drying and dry blending. Usefulness of the product is further limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve, especially in the laboratories where the products are to be utilized in diagnostic assay. This condition is exemplified by the fact that most commercial freeze-dried controlled sera (reference serum) lists the acceptable bottle-to-bottle variation of enzyme constituents at .+-.10% of the mean. Generally, the reconstituted freeze-dried urease solutions have a stability of about 24 hours to 5 days at room temperature conditions. Their use is then limited by such short shelf life.
The present invention is uniquely designed so that the enzyme solution, although containing labile ingredients in a liquid reagent, is effectively "stabilized" thereby controlling the activity of the labile ingredients in the liquid solution. The means of stability insures long-term stability in a liquid media. Moreover, close tolerance control can be achieved in the manufacturing of a high quality product which eliminates the inconvenience of the rigid package size, the high cost of packaging and freeze-drying, and reagent waste.